Methods and systems for production of dna libraries directly from a stool sample for 16s metagenomics next generation sequencing

ABSTRACT

Disclosed are methods for preparing a DNA library directly from a stool sample. The method comprises applying the stool sample directly to a buffer, heating and cooling the buffer, separating a supernatant within the buffer from a precipitate using centrifugation, and transferring the supernatant into a first reaction vessel containing a first reagent mixture to yield a first reaction mixture. The method also comprises subjecting the first reaction mixture to a first PCR protocol, purifying amplicons within the first reaction vessel through a first purification procedure to yield a purified target amplicon solution, transferring the purified target amplicon solution to a second reaction vessel comprising a second reagent mixture to yield a second reaction mixture, and subjecting the second reaction mixture to a second PCR protocol. The method further comprises purifying index-tagged amplicons within the second reaction vessel through a second purification procedure to yield the DNA library.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of priority to U.S. ProvisionalPatent Application No. 62/955,711, filed on Dec. 31, 2019, the contentof which is hereby incorporated by reference in its entirety.

TECHNICAL FIELD

The present disclosure relates generally to the field of samplepreparation for genetic sequencing; more specifically, to methods,compositions, and kits for the production of deoxyribonucleic acid (DNA)libraries directly from a stool sample for next generation sequencing.

BACKGROUND

Metagenomics is a molecular tool used to analyze DNA sequences obtainedfrom environmental samples in order to study the community ofmicroorganisms present. The human intestinal tract has a variety ofmicrobial communities that play an important role in the health of thehuman host. Advances in high-throughput DNA next generation sequencing(NGS) technology have enabled researchers to more quickly andefficiently reveal changes in the composition and function of gutmicrobes, which are often associated with various diseases or diseasestates, including cancer, AIDS and other illnesses. In addition,metagenomic analysis provides a better understanding of normal gutmicrobiome and its relationship to various exogenous and endogenous hostfactors. Recent studies have shown that microbiome distribution or thedistribution of bacterial species within a person's gut can be used notonly for early diagnosis and prognosis of disease, but also forindividualized treatment options in everyday medical practice.

The use of DNA NGS technology to analyze microbial populations isgenerally divided into the following steps: 1) bacterial samplecollection, 2) DNA isolation or extraction, 3) DNA library preparation,4) DNA library sequencing, and 5) data analysis.

Human gut bacterial DNA extraction is a tedious and very unpleasant jobbecause it often starts off with collecting, weighing, and homogenizinga sample containing human feces. For example, FIG. 1 shows certaininitial steps of a conventional method of preparing DNA libraries from astool sample. These initial steps are often laborious and time-consumingand include stool sample collection, sample weighing, samplehomogenization, and extracting the DNA from the homogenized samplethrough enzymatic digestion, centrifugation, and numerous washing steps.Such conventional methods also require multiple reagents and buffers andexpensive one-time-use spin columns. Such conventional methods are alsosusceptible to high risks of clinician error.

Therefore, a solution is needed which reduces the number of initialoperational steps needed to prepare DNA libraries (e.g., 16Smetagenomics DNA libraries) from a stool sample for next generationsequencing yet maintain or improve the quantity and quality of targetsequence yields compared to conventional methods. Such a solution shouldbe cost-effective compared to conventional methods, require less time,and should lessen the risk of clinician or operator error.

SUMMARY

Disclosed herein are methods, compositions, and kits for the preparationof DNA libraries directly from a stool sample for downstreamnext-generation sequencing. In one embodiment, a method comprisesapplying a stool sample directly to a buffer solution and heating andcooling the buffer solution containing the stool sample. In someembodiments, applying the stool sample directly to the buffer solutioncan further comprise applying between about 3 mg to 10 mg of the stoolsample to about 100 μL of the buffer solution

In certain embodiments, the buffer solution can comprise Tris-HCl, EDTA,and polyacrylic acid. Moreover, heating and cooling the buffer solutioncan comprise heating the buffer solution containing the stool sampleabove a temperature threshold and subsequently cooling the buffersolution containing the stool sample to room temperature.

The method can further comprise separating a supernatant within thebuffer solution containing the stool sample from a precipitate usingcentrifugation. The method can also comprise transferring an aliquot ofthe supernatant into a first reaction vessel containing a first reagentmixture to yield a first reaction mixture. Transferring the aliquot ofthe supernatant into the first reagent mixture can further comprisetransferring about 2 μL of the supernatant into the first reagentmixture in the first reaction vessel.

In some embodiments, the first reagent mixture can comprise Taq DNApolymerase, dNTPs, a primer pool comprising a plurality of forwardprimers and reverse primers, a cofactor, a nonionic surfactant, agelatin solution, a glycerol solution, and a reagent buffer.

In some embodiments, the primer pool can comprise a plurality of 16Sforward primers and 16S reverse primers for targeting variable regionsV3 and V4 of the 16S ribosomal ribonucleic acid (rRNA) gene. Moreover,the reagent buffer can comprise a Tris-HCl buffer solution and apotassium chloride (KCl) buffer solution. In addition, the cofactor canbe magnesium chloride (MgCl₂) and the nonionic surfactant can be apolysorbate 20 solution

The method can also comprise subjecting the first reaction mixture inthe first reaction vessel to a first polymerase chain reaction (PCR)protocol. The first PCR protocol can comprise the steps of: (i) heatingthe first reaction mixture at a first temperature to activate the TaqDNA polymerase in an activation step, (ii) further heating the firstreaction mixture at a second temperature to denature nucleic acidswithin the first reaction mixture, (iii) lowering the temperature to athird temperature to allow for annealing and extension, (iv) repeatingsteps (ii) and (iii) for at least 4 more cycles, (v) further heating thefirst reaction mixture at a fourth temperature to further denaturenucleic acids within the first reaction mixture, (vi) lowering thetemperature to a fifth temperature to allow for annealing and extension,(vii) repeating steps (v) and (vi) for at least 24 more cycles, and(viii) holding the amplified first reaction mixture within the firstreaction vessel at a holding temperature.

The method can also comprise purifying the first reaction mixture withinthe first reaction vessel through a first purification procedure. Thefirst purification procedure can comprise: (a) introducing a magneticbead suspension to the first reaction vessel, (b) incubating a mixturewithin the first reaction vessel comprising the magnetic bead suspensionat room temperature for an incubation period to allow amplicons to bindto beads within the magnetic bead suspension, (c) collecting andimmobilizing the amplicon-bound magnetic beads to at least one innersurface of the first reaction vessel by placing at least one outersurface of the first reaction vessel in proximity to a magnet, (d)removing and discarding a supernatant from the first reaction vesselwhile the amplicon-bound magnetic beads are immobilized to the at leastone inner surface of the first reaction vessel by the magnet, (e)introducing an ethanol wash solution to the first reaction vesselcomprising the amplicon-bound magnetic beads, (f) removing anddiscarding a supernatant from the first reaction vessel while theamplicon-bound magnetic beads are immobilized to the at least one innersurface of the first reaction vessel by the magnet, (g) introducingwater to the first reaction vessel to elute amplicons bound to themagnetic beads, (h) removing a first amplicon-containing supernatantfrom the first reaction vessel after the introduction of water while themagnetic beads are immobilized to the at least one inner surface of thefirst reaction vessel by the magnet, (i) adding the firstamplicon-containing supernatant from step (h) to an intermediaryreaction vessel and repeating steps (a) through (g) using contentswithin the intermediary reaction vessel, and (j) removing a secondamplicon-containing supernatant from the intermediary reaction vesselafter the introduction of the water while the magnetic beads areimmobilized to at least one inner surface of the intermediary reactionvessel by the magnet. The second amplicon-containing supernatant removedis a purified target amplicon solution that can be further amplified andpurified through subsequent steps of the method.

In some embodiments, the first reaction vessel can be a standalonereaction tube or container such as a standalone PCR tube. In otherembodiments, the reaction vessel can be a well of a multi-well plate.The magnet can be part of a magnetic separation rack or platform.

In certain embodiments, collecting and immobilizing the amplicon-boundmagnetic beads can further comprise positioning the at least one outersurface of the first reaction vessel next to the magnet and repeatedlymoving the first reaction vessel away from the magnet and back next tothe magnet.

The method can further comprise transferring the purified targetamplicon solution to a second reaction vessel comprising a secondreagent mixture to yield a second reaction mixture.

In some embodiments, the second reagent mixture can comprise Taq DNApolymerase, dNTPs, a plurality of sequencing index adapters, a cofactor,a nonionic surfactant, a gelatin solution, a glycerol solution, and areagent buffer.

The method can further comprise subjecting the second reaction mixturein the second reaction vessel to a second PCR protocol. The second PCRprotocol can comprise the steps of: (i) heating the second reactionmixture at a first temperature to activate the Taq DNA polymerase in anactivation step; (ii) further heating the second reaction mixture at asecond temperature to denature nucleic acids within the reactionmixture; (iii) lowering the temperature to a third temperature to allowfor annealing and extension, (iv) adjusting the temperature to a fourthtemperature to allow for additional extension, (v) repeating steps (ii)through (iv) for at least 7 more cycles, (vi) further heating the secondreaction mixture at a fifth temperature to allow for further extension,and (vii) holding the amplified second reaction mixture within thesecond reaction vessel at a holding temperature.

The method can further comprise purifying index-tagged amplicons withinthe second reaction vessel through a second purification procedure toyield a purified index-tagged DNA library. The second purificationprocedure can comprise (a) introducing additional instances of themagnetic bead suspension to the second reaction vessel, (b) incubating amixture within the second reaction vessel comprising the magnetic beadsuspension at room temperature for an incubation period to allowamplicons to bind to beads within the magnetic bead suspension, (c)collecting and immobilizing the amplicon-bound magnetic beads to atleast one inner surface of the second reaction vessel by placing atleast one outer surface of the second reaction vessel in proximity to amagnet, (d) removing and discarding a supernatant from the secondreaction vessel while the amplicon-bound magnetic beads are immobilizedto the at least one inner surface of the second reaction vessel by themagnet, (e) introducing an ethanol wash solution to the second reactionvessel comprising the amplicon-bound magnetic beads, (f) removing anddiscarding a supernatant from the second reaction vessel while theamplicon-bound magnetic beads are immobilized to the at least one innersurface of the second reaction vessel by the magnet, (g) introducingwater to the second reaction vessel to elute amplicons bound to themagnetic beads, (h) removing a first amplicon-containing supernatantfrom the second reaction vessel after the introduction of the waterwhile the magnetic beads are immobilized to the at least one innersurface of the second reaction vessel by the magnet, (i) adding thefirst amplicon-containing supernatant from step (h) to anotherintermediary reaction vessel and repeating steps (a) through (g) usingcontents within the other intermediary reaction vessel, and (j) removinga second amplicon-containing supernatant from the other intermediaryreaction vessel after the introduction of the water while the magneticbeads are immobilized to at least one inner surface of the otherintermediary reaction vessel by the magnet.

The second amplicon-containing supernatant removed is the purified DNAlibrary that can be sequenced using a next-generation sequencingprotocol. For example, the DNA library generated from this method can besequenced using an Illumina® NGS protocol, an Ion PGM® protocol, aSOLiD® NGS protocol, or a combination thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates certain steps of a method known in the art forpreparing DNA libraries from a stool sample.

FIG. 2 illustrates an embodiment of an improved method of preparing aDNA library directly from a stool sample.

FIG. 3 illustrates the introduction of a stool sample into a buffersolution using a swab.

FIG. 4 illustrates certain initial steps of the method for preparing aDNA library directly from a stool sample.

FIG. 5 illustrates an embodiment of a first purification procedure usingmagnetic beads.

FIG. 6A illustrates an embodiment of a magnetic separation rack used aspart of a purification procedure.

FIG. 6B illustrates another embodiment of a magnetic separation rackused as part of the purification procedure.

FIG. 6C illustrates an embodiment of a multi-well plate positioned onthe magnetic separation rack shown in FIG. 6B.

FIG. 6D is a black-and-white image illustrating an embodiment of amulti-well plate having magnetic beads immobilized to an inner sidesurface of wells of the well plate.

FIG. 7 illustrates additional steps of the method for preparing a DNAlibrary directly from a stool sample.

FIG. 8 illustrates an embodiment of a second purification procedureusing magnetic beads.

FIG. 9 is bioanalyzer trace showing the size distribution of a 16S DNAlibrary prepared directly from a stool sample using the method disclosedherein.

FIGS. 10A to 10C illustrate comparisons of 16S DNA libraries preparedfrom three different stool samples using traditional DNA extractionmethods and 16S DNA libraries prepared from the same three stool samplesusing the method disclosed herein

DETAILED DESCRIPTION

Disclosed herein are methods, compositions, and kits for the preparationof DNA libraries directly from a stool sample for metagenomics nextgeneration sequencing. A DNA library is a collection of genomic DNAsequences of interest obtained from a tissue sample of an organism. Themethods, compositions, and kits disclosed herein are optimized for thepreparation of DNA libraries for further downstream next-generationsequencing (NGS) for clinical diagnosis and research. For example, theDNA libraries prepared using the methods, compositions, and kitsdisclosed herein can be used to identify or diagnose pathogenicbacteria. The DNA libraries generated from the methods, compositions,and kits disclosed herein can be used with any number of NGS platforms,including platforms requiring immobilization of DNA fragments onto asolid support, cyclic sequencing reactions using automated devices, anddetection of sequences using imaging or semiconductor technologies. Forexample, the DNA libraries generated from the methods, compositions, andkits disclosed herein can be used with an Illumina® sequencing bysynthesis (SBS) NGS platform (e.g., an Illumina MiniSeq®, MiSeq®, orNextSeq® system) distributed by Illumina, Inc., an Ion Personal GenomeMachine® (PGM) system distributed by Thermo Fisher Scientific Inc., aSOLiD® NGS system distributed by Thermo Fisher Scientific Inc., or acombination thereof.

FIG. 2 illustrates an embodiment of an improved method 200 of preparinga DNA library directly from a stool sample 300 (see FIG. 3). The stoolsample 300 can be a fecal sample obtained from a human subject (i.e.,human fecal matter) or an animal subject (i.e., animal fecal matter). Insome embodiments, the stool sample 300 can comprise trace amounts ofcontaminants such as dirt, mud, hair, or other environmentalcontaminants.

The method 200 can also be used to prepare a DNA library directly from asample of vomit. In further embodiments, the method 200 can also be usedto prepare a DNA library directly from an environmental sample such as asample of mud or a slurry comprising sand and liquid.

The method 200 can comprise applying the stool sample 300 to a buffersolution 302 (see FIG. 3) in operation 202. For example, the stoolsample 300 can be dropped or stirred into the buffer solution 302. Inother embodiments, the stool sample 300 can be smeared along an innersurface of a sample collection tube containing the buffer solution 302.

Applying the stool sample 300 directly to the buffer solution 302further comprises applying between about 3 mg to about 10 mg of thestool sample 300 to between about 100 μL to about 150 μL of the buffersolution 302. In other embodiments, applying the stool sample 300directly to the buffer solution 302 can comprise applying about 5 mg ofthe stool sample 300 to about 100 μL of the buffer solution 302.

The buffer solution 302 can comprise atris(hydroxymethyl)aminomethane-hydrochloric acid (Tris-HCl) buffer,ethylenediaminetetraacetic acid (EDTA), and polyacrylic acid.

Presented in Table 1 below is an example formulation of the buffersolution 302:

TABLE 1 EXAMPLE BUFFER SOLUTION Solution Component ConcentrationTris-HCl, pH 8.0 30.0.0 mM EDTA 5 mM Polyacrylic acid 0.10% (v/v)

The method 200 can further comprise heating and cooling the buffersolution 302 containing the stool sample 300 in operation 204. Operation204 can also comprise homogenizing the buffer solution 302 prior toheating and cooling the buffer solution 302 containing the stool sample300. For example, the method 200 can also comprise homogenizing thebuffer solution 302 containing the stool sample 300 using a vortex mixeror shaker. In other embodiments, the stool sample 300 can be stirredinto the buffer solution 302 using a stirring rod or stirrer.

The method 200 can further comprise heating the buffer solution 302containing the stool sample 300 above a threshold temperature andsubsequently cooling the buffer solution 302 containing the stool sample300 to room temperature in operation 204. In some embodiments, thethreshold temperature can be between about 90° C. to about 100° C. Morespecifically, the threshold temperature can be about 95° C.

One unexpected discovery made by the applicant is that the buffersolution 302 having the composition disclosed herein combined with theheating and cooling steps disclosed herein are effective in facilitatingthe breakdown of the stool sample 300 without unduly interfering withthe quality of the nucleic acids for further downstream processing.

The method 200 can also comprise separating a supernatant 400 (see FIG.4) within the buffer solution 302 containing the stool sample 300 from aprecipitate using centrifugation in operation 206. The buffer solution302 containing the stool sample 300 can be centrifuged when the solutionhas reached room temperature. Operation 206 can also comprisetransferring an aliquot of the supernatant 400 into a first reactionvessel 402 containing a first reagent mixture 404 to yield a firstreaction mixture 406 (see, FIG. 4).

In some embodiments, transferring the aliquot of the supernatant 400into the first reaction vessel 402 containing the first reagent mixture404 can comprise transferring about 2 μL of the supernatant 400 into thefirst reaction vessel 402 containing about 18 μL of the first reagentmixture 404. In other embodiments, transferring the aliquot of thesupernatant 400 into the first reaction vessel 402 containing the firstreagent mixture 404 can comprise transferring between about 2 μL toabout 5 μL of the supernatant 400 into the first reaction vessel 402containing the first reagent mixture 404. The aliquot of the supernatant400 can be transferred using a pipette such as a fixed-volumemicropipette or an adjustable-volume micropipette.

The first reagent mixture 404 can comprise a reagent solution and aprimer pool comprising a plurality of forward primers and reverseprimers for the target sequences of interest. The reagent solution cancomprise a DNA polymerase, a plurality of dNTPs, a cofactor, a nonionicsurfactant, a gelatin solution, a glycerol solution, and one or morereagent buffers.

In some embodiments, the one or more reagent buffers can comprise atris(hydroxymethyl)aminomethane (Tris) buffer solution (e.g., aTris-hydrochloric acid (HCl) buffer solution), a potassium chloride(KCl) buffer solution, or a combination thereof.

In some embodiments, the cofactor or cofactor solution can be amagnesium chloride (MgCl₂) solution. The nonionic surfactant can be apolysorbate solution (e.g., a polysorbate 20 solution). Morespecifically, the nonionic surfactant can be a Tween® 20 surfactantdistributed by Sigma-Aldrich, Inc., a Montanox™ 20 surfactantdistributed by SEPPIC S.A., or an Alkest® 20 surfactant distributed byOxiteno S.A.

In some embodiments, the DNA polymerase can be a thermostable DNApolymerase such as a Taq DNA polymerase. For example, the Taq DNApolymerase can be a Taq DNA polymerase provided by Thermo FisherScientific Inc.

The concentration of the Tris-HCl buffer solution can be between about60.0 mM and about 90.0 mM. The pH of the Tris-HCl buffer can be at a pHof about 8.0. The concentration of the KCl buffer solution can bebetween about 100.0 mM and about 150.0 mM. The concentration of theMgCl₂ solution can be between about 2.0 mM and about 5.0 mM. Thepolysorbate 20 solution can be between about 0.01% (v/v) and about 0.30%(v/v) of the total volume of the reagent solution. The glycerol solutioncan be between about 10.0% (v/v) and about 30.0% (v/v) of the totalvolume of the reagent solution. The gelatin solution can be betweenabout 0.01% (v/v) and about 0.80% (v/v) of the total volume of thereagent solution.

As contemplated by this disclosure and as will be appreciated by one ofordinary skill in the art, the reagent solution can be made at differentconcentrations and provided as 1× to 5× (e.g., 1×, 2×, 3×, 4×, or 5×)master mixes. Presented in Table 2 below is an example formulation of areagent solution:

TABLE 2 EXAMPLE COMPOSITION OF 2X REAGENT SOLUTION Solution ComponentConcentration Taq DNA polymerase 0.5 Units/μL-0.8 Units/μL dNTPs (dATP,dCTP, dGTP, and dTTP) 1.0 mM-3.0 mM MgCl₂ 2.0 mM-5.0 mM Polysorbate 20   0.01%-0.30% (v/v) Glycerol    10.0%-30.0% (v/v) Gelatin   0.01%-0.80% (v/v) KCl 100.0 mM-150.0 mM Tris-HCl, pH 8.0 60.0 mM-90.0mM

In some embodiments, 90% (v/v) of the first reaction mixture 406 can bethe first reagent mixture 404 and 10% (v/v) of the first reactionmixture 406 can be the supernatant 400. More specifically, the firstreaction mixture 406 can be comprised of 50% (v/v) 2× reagent solution,20% (v/v) 20× primer pool solution, 20% (v/v) deionized water 410, and10% (v/v) supernatant 400. Presented in Table 3 below is an exampleformulation of 20 μL of the first reaction mixture 406:

TABLE 3 EXAMPLE FIRST REACTION MIXTURE COMPOSITION Percentage of DropletComponent Volume Total Volume 2X Reagent Solution 10 μL  50% 20X 16SPrimer Pool 4 μL 20% Deionized water 4 μL 20% Supernatant containingnucleic acids 2 μL 10% TOTAL: 20 μL  100% 

In other embodiments, the first reaction mixture 406 can be comprised of50% (v/v) 2× reagent solution, 5% (v/v) 20× primer pool solution, 35%(v/v) deionized water 410, and 10% (v/v) supernatant 400. Presented inTable 4 below is another example formulation of 20 μL of the firstreaction mixture 406:

TABLE 4 EXAMPLE FIRST REACTION MIXTURE COMPOSITION Percentage of DropletComponent Volume Total Volume 2X Reagent Solution 10 μL  50% 20X 16SPrimer Pool 1 μL  5% Deionized water 7 μL 35% Supernatant containingnucleic acids 2 μL 10% TOTAL: 20 μL  100% 

In some embodiments, the primer pool can comprise a plurality of forwardprimers and reverse primers. For example, the primer pool can comprise aplurality of 16S forward primers and 16S reverse primers for targetingvariable regions V3 and V4 of the 16S ribosomal ribonucleic acid (rRNA)gene. When such 16S primers are used, the DNA library generated can beconsidered a 16S DNA library. In other embodiments, the primer pool cancomprise a plurality of forward primers and reverse primers targetingother regions of interest.

The method 200 can further comprise subjecting the first reactionmixture 406 in the first reaction vessel 402 to a first polymerase chainreaction (PCR) protocol in step 208. The first PCR protocol can comprise(i) heating the first reaction mixture 406 at a first temperature toactivate the DNA polymerase in an activation step. The first PCRprotocol can also comprise (ii) further heating the first reactionmixture 406 at a second temperature to denature nucleic acids (templateDNA) within the first reaction mixture 406 in a denaturation step. Thefirst PCR protocol can further comprise (iii) lowering the temperatureto a third temperature to allow for annealing of the primers to thetemplate DNA and extension or elongation of the annealed primers by theDNA polymerase. The first PCR protocol can also comprise (iv) repeatingthe (ii) denaturation and (iii) annealing and extension steps for atleast 4 more cycles (so 5 cycles total). The first PCR protocol can alsocomprise (v) further heating the first reaction mixture at a fourthtemperature to further denature nucleic acids within the first reactionmixture. The first PCR protocol can further comprise (vi) lowering thetemperature to a fifth temperature to allow for further annealing andextension. The first PCR protocol can also comprise (vii) repeating the(v) denaturation and (vi) annealing and extension steps for at least 24more cycles (so 25 cycles total).

In other embodiments, the (v) denaturation and (vi) annealing andextension steps can be repeated for between 25 cycles and 30 cycles. Thefirst PCR protocol can also comprise holding the amplified firstreaction mixture 406 within the first reaction vessel 402 at a holdingtemperature.

In some embodiments, the first temperature of the first PCR protocol canbe about 95° C. (i.e., the activation temperature can be about 95° C.),the second temperature of the first PCR protocol can also be about 95°C. (i.e., the denaturation temperature can be about 95° C.), the thirdtemperature of the first PCR protocol can be about 60° C. (i.e., theannealing and extension temperature can be about 60° C.), the fourthtemperature of the first PCR protocol can be about 95° C. (i.e., thedenaturation temperature can be about 95° C.), the fifth temperature ofthe first PCR protocol can be about 72° C. (i.e., the annealing andextension temperature can be about 72° C.), and the holding temperaturecan be about 8° C.

Presented in Table 5 below is an example first PCR protocol:

TABLE 5 EXAMPLE FIRST PCR PROTOCOL Enzyme Annealing and Annealing andActivation Denaturation Extension Denaturation Extension Cooling StepStep Steps Step Steps Step Temp: ~95° C. Temp: ~95° C. Temp: ~60° C.Temp: ~95° C. Temp: ~72° C. Temp: ~8° C. Time: ~15 min. Time: ~1 min.Time: ~6 min. Time: ~30 sec. Time: ~3 min. Hold 5 Cycles 25 Cycles

After undergoing the aforementioned first PCR protocol, the firstreaction mixture 406 can be purified to obtain or collect the amplifiedsequences.

The method 200 can further comprise purifying or isolating the ampliconswithin the first reaction vessel 402 through a first purificationprocedure 500 (see, for example, FIG. 5) in operation 210. The firstpurification procedure 500 can comprise purifying or isolating theamplicons using magnetic beads 504 (see, for example, FIG. 5). The firstpurification procedure 500 can further comprise subjecting theamplicon-bound magnetic beads 507 to multiple ethanol washes and elutingthe target amplicons using water to yield a purified target ampliconsolution 518. The first purification procedure 500 will be discussed inmore detail in the following sections.

The method 200 can further comprise transferring the purified targetamplicon solution 518 to a second reaction vessel 700 comprising asecond reagent mixture 702 to yield a second reaction mixture 704 (see,for example, FIG. 7) in operation 212.

In some embodiments, transferring the aliquot of the purified targetamplicon solution 518 into the second reaction vessel 700 containing thesecond reagent mixture 702 can comprise transferring about 10.5 μL ofthe purified target amplicon solution 518 into the second reactionvessel 700 containing about 14.5 μL of the second reagent mixture 702.The aliquot of the purified target amplicon solution 518 can betransferred using a pipette such as a fixed-volume micropipette or anadjustable-volume micropipette.

The second reagent mixture 702 can comprise a reagent solution and anindex primer pool comprising a plurality of index adapteroligonucleotides or index adapters. The reagent solution can be the samereagent solution disclosed in the previous sections (see, e.g., Table2). For example, the reagent solution can comprise a Taq DNA polymerase,a plurality of dNTPs, a cofactor, a nonionic surfactant, a gelatinsolution, a glycerol solution, and one or more reagent buffers.

In some embodiments, about 58% (v/v) of the second reaction mixture 704can be the second reagent mixture 702 and about 42% (v/v) of the secondreaction mixture 704 can be the purified target amplicon solution 518.More specifically, the second reaction mixture 704 can be comprised of50% (v/v) 2× reagent solution, 8% (v/v) index primer pool solution, and42% (v/v) purified target amplicon solution 518. Presented in Table 6below is an example formulation of 25 μL of the second reaction mixture704:

TABLE 6 EXAMPLE SECOND REACTION MIXTURE COMPOSITION Percentage ofDroplet Component Volume Total Volume 2X Reagent Solution 12.5 μL 50%Nextera ® XT Index 1 Primers 1 μL  4% Nextera ® XT Index 2 Primers 1 μL 4% Purified target amplicon solution 10.5 μL 42% TOTAL: 25 μL 100% 

Presented in Table 7 below is another example formulation of 12.5 μL ofthe second reaction mixture 704:

TABLE 7 EXAMPLE SECOND REACTION MIXTURE COMPOSITION Percentage ofDroplet Component Volume Total Volume 2X Reagent Solution 6.25 μL 50%Nextera ® XT Index 1 Primers 0.5 μL  4% Nextera ® XT Index 2 Primers 0.5μL  4% Purified target amplicon solution 5.25 μL 42% TOTAL: 12.5 μL100% 

In some embodiments, the index primer pool can comprise a plurality offorward index primers and reverse index primers. For example, the indexprimer pool can comprise a plurality of forward index adapteroligonucleotides or forward index adapters and a plurality reverse indexadapter oligonucleotides or reverse index adapters. The index adapterscan be annealed or added to the ends of the amplified target sequences(or target amplicons) after the completion of the second PCR protocol.The index adapters when added to the ends of the target amplicons canact as barcodes or unique identifiers to identify the target ampliconswhen the DNA library is being sequenced using next-generationsequencing. Once the target amplicons are tagged with the indexadapters, the DNA library can be considered ready for sequencing usingnext-generation sequencing systems such as the Illumina MiSeq® system.Different pairs of index adapters can also be used to allow multiplepooled samples to be sequenced together in a single high-throughputnext-generation sequencing run.

The method 200 can further comprise subjecting the second reactionmixture 702 in the second reaction vessel 700 to a second PCR protocolin operation 214. The second PCR protocol can be a limited-cycleprotocol for adding index adapters to the ends of the target amplicons(i.e., index-tagging the target amplicons) and amplifying theindex-tagged target amplicons. For example, when the sequence ofinterest is the 16S rRNA gene, the second PCR protocol can be alimited-cycle protocol for index-tagging the 16S amplicons andamplifying the index-tagged 16S amplicons.

The second PCR protocol can comprise (i) heating the second reactionmixture 702 at a first temperature to activate the DNA polymerase in anactivation step. The second PCR protocol can also comprise (ii) furtherheating the second reaction mixture 702 at a second temperature todenature nucleic acids within the second reaction mixture 702 in adenaturation step.

The second PCR protocol can further comprise (iii) lowering thetemperature to a third temperature to allow for annealing of the indexprimers to the target amplicons and extension or elongation of theannealed index primers by the DNA polymerase. The second PCR protocolcan also comprise (iv) further heating at a fourth temperature to allowfor further extension. The second PCR protocol can also comprise (v)repeating the (ii) denaturation, (iii) annealing and extension, and (iv)further extension steps for at least 7 more cycles (so 8 cycles total).In other embodiments, steps (ii) through (v) can be repeated for between8 cycles and 10 cycles.

The second PCR protocol can also comprise (vi) further heating thesecond reaction mixture 702 at a fifth temperature to allow for furtherextension. The second PCR protocol can also comprise holding theindex-tagged amplicons within the second reaction vessel 700 at aholding temperature.

In some embodiments, the first temperature of the second PCR protocolcan be about 95° C. (i.e., the activation temperature can be about 95°C.), the second temperature of the second PCR protocol can also be about95° C. (i.e., the denaturation temperature can be about 95° C.), thethird temperature of the second PCR protocol can be about 66° C. (i.e.,the annealing and extension temperature can be about 66° C.), the fourthtemperature of the second PCR protocol can be about 72° C. (i.e., thefurther extension temperature can be about 72° C.), the fifthtemperature of the second PCR protocol can be about 72° C. (i.e., thefinal extension temperature can be about 72° C.), and the holdingtemperature can be about 4° C.

Presented in Table 8 below is an example second PCR protocol:

TABLE 8 EXAMPLE SECOND PCR PROTOCOL Enzyme Annealing and FinalActivation Denaturation Extension Extension Extension Cooling Step StepSteps Step Step Step Temp: ~95° C. Temp: ~95° C. Temp: ~66° C. Temp:~72° C. Temp: ~72° C. Temp: ~4° C. Time: ~2 min. Time: ~30 sec. Time:~30 sec. Time: ~60 sec. Time: ~5 min. Hold 8 Cycles

After undergoing the aforementioned second PCR protocol, theindex-tagged amplicons can be purified to obtain an index-tagged DNAlibrary ready for next generating sequencing.

The method 200 can further comprise purifying or isolating theindex-tagged amplicons within the second reaction vessel 700 through asecond purification procedure 800 (see, for example, FIG. 8) inoperation 216. The second purification procedure 800 can comprisepurifying or isolating the index-tagged amplicons using magnetic beads504 (see, for example, FIG. 8). The second purification procedure 800can further comprise subjecting the amplicon-bound magnetic beads 804 tomultiple ethanol washes and eluting the index-tagged amplicons usingwater to yield a purified index-tagged DNA library 810. The secondpurification procedure 800 will be discussed in more detail in thefollowing sections.

The index-tagged DNA library 810 generated from this method 200 can besequenced using (but not limited to) an Illumina® NGS protocol, an IonPGM® protocol, a SOLiD® NGS protocol, or a combination thereof.

One unexpected discovery made by the applicant is that the first PCRprotocol disclosed herein is effective in amplifying target sequencesfrom the buffer solution comprising the stool sample. Moreover, theamplified sequences obtained from the aforementioned first PCR protocolare uniform and high in quantity.

Another unexpected discovery is that the purification proceduresdisclosed herein (e.g., the first purification procedure 500 and thesecond purification procedure 800) are effective in purifying the targetamplicons after the first PCR protocol and the index-tagged targetamplicons after the second PCR protocol. Moreover, the DNA library(e.g., a 16S DNA library) resulting from such purification proceduresare of high-quality and ready for sequencing using NGS protocols.

Yet another unexpected discovery made by the applicant is that DNAlibraries (e.g., 16S DNA libraries) could be prepared from much smalleramounts of stool sample using the method 200 disclosed herein thantraditional extraction methods.

FIG. 3 illustrates that the stool sample 300 can be applied using a swab302 or pick. In some embodiments, the swab 304 can be a traditionalcotton swab or Q-tip. In other embodiments, the swab 304 can comprise apolymeric swab head and handle. For example, the swab 304 can comprise apolyester fabric head and a polypropylene handle. In furtherembodiments, the swab 304 can comprise a swab head made of Teflon coatedfiberglass.

In some embodiments, the swab 302 carrying the stool sample 300 can dropthe stool sample 300 into the buffer solution 302. In other embodiments,the stool sample 300 can be stirred into the buffer solution 302 usingthe swab 302.

FIG. 4 illustrates that the supernatant 400 within the buffer solution302 containing the stool sample 300 can be separated from a precipitateusing centrifugation. The buffer solution 302 containing the stoolsample 300 can be centrifuged when the solution reaches roomtemperature. FIG. 4 also illustrates that an aliquot of the supernatant400 can be transferred into a first reaction vessel 402 containing afirst reagent mixture 404 to yield a first reaction mixture 406.

In some embodiments, the first reaction vessel 402 can be a single PCRreaction tube 408 or vessel. In other embodiments, the first reactionvessel 402 can be one well 410 of a multi-well plate 412 (e.g., amulti-well PCR plate), such as a 96-well plate or a 384-well plate.

In some embodiments, transferring the aliquot of the supernatant 400into the first reaction vessel 402 containing the first reagent mixture404 can comprise transferring about 2 μL of the supernatant 400 into thefirst reaction vessel 402 containing about 18 μL of the first reagentmixture 404. In other embodiments, transferring the aliquot of thesupernatant 400 into the first reaction vessel 402 containing the firstreagent mixture 404 can comprise transferring between about 2 μL toabout 5 μL of the supernatant 400 into the first reaction vessel 402containing the first reagent mixture 404. The aliquot of the supernatant400 can be transferred using a pipette such as a fixed-volumemicropipette or an adjustable-volume micropipette.

The first reagent mixture 404 can comprise a reagent solution and aprimer pool comprising a plurality of forward primers and reverseprimers for the target sequences of interest. The reagent solution cancomprise a DNA polymerase, a plurality of dNTPs, a cofactor, a nonionicsurfactant, a gelatin solution, a glycerol solution, and one or morereagent buffers.

FIG. 5 illustrates an embodiment of the first purification procedure500. The first purification procedure 500 can comprise introducing amagnetic bead suspension 502 to the first reaction vessel 402. Themagnetic bead suspension 502 can comprise magnetic beads 504 configuredto allow the target amplicons within the amplified first reactionmixture 406 to selectively bind to surfaces of the magnetic beads 504.For example, the magnetic bead suspension 502 can be AMPure® beadsmanufactured by Beckman Coulter, Inc.

In some embodiments, the volume of the magnetic bead suspension 502added is anywhere between 1× to 1.8× the volume of the first reactionmixture 406 within the first reaction vessel 402. For example, 36 μL ofthe magnetic bead suspension 502 can be added to 20 μL of the firstreaction mixture 406 within the first reaction vessel 402.

The first purification procedure 500 can also comprise incubating amixture 506 within the first reaction vessel 402 comprising both theamplified first reaction mixture 406 and the magnetic bead suspension502 at room temperature (e.g., between about 20° C. to about 25° C.) foran incubation period (e.g., between about 5 minutes to 10 minutes) toallow the target amplicons to bind to the magnetic beads 504.

The first purification procedure 500 can also comprise collecting andimmobilizing amplicon-bound magnetic beads 507 to at least one innersurface of the first reaction vessel 402 by placing at least one outersurface of the first reaction vessel 402 in proximity to a magnet 508.The first purification procedure 500 can further comprise positioningthe at least one outer surface of the first reaction vessel 402 inproximity to the magnet 508 and then repeatedly moving the firstreaction vessel 402 away from the magnet 508 and bringing the at leastone outer surface of the reaction vessel 402 back next to the magnet508.

In some embodiments, the magnet 508 can be a permanent magnet. Forexample, the magnet 508 can be a neodymium iron boron (NdFeB) permanentmagnet. The magnet 508 can be incorporated into or embedded within amagnetic separation rack or platform (see, e.g., FIGS. 6A and 6B).

The first purification procedure 500 can also comprise removing anddiscarding a supernatant from the first reaction vessel 402 while theamplicon-bound magnetic beads 507 are immobilized to the at least oneinner surface of the first reaction vessel 402 by the magnet 508.Removing and discarding the supernatant can comprise using amicropipette to aspirate the supernatant from the first reaction vessel402 into the pipette tip and expelling the supernatant to discard thesupernatant.

The first purification procedure 500 can also comprise introducing anethanol wash solution 510 to the first reaction vessel 402 containingthe amplicon-bound magnetic beads 507. For example, the ethanol washsolution 510 can be a 70% (v/v) ethanol or isopropyl alcohol solution.The first purification procedure 500 can comprise introducing betweenabout 50 μL to about 125 μL of the ethanol wash solution 510 to thefirst reaction vessel 402 containing the amplicon-bound magnetic beads507. One objective of the ethanol wash step is to remove excess saltsfrom buffers added to the first reaction vessel 402 in previous steps ofthe method 200.

The first purification procedure 500 can further comprise removing anddiscarding a supernatant comprising primarily of the ethanol washsolution 510 from the first reaction vessel 402 while the amplicon-boundmagnetic beads 507 are immobilized to the at least one inner surface ofthe first reaction vessel 402 by the magnet 508. The first purificationprocedure can also comprise drying (e.g., air drying) the first reactionvessel 402 after each ethanol wash to evaporate the ethanol left over.The ethanol wash steps can be repeated one or more times in succession.For example, the ethanol wash steps can be performed twice before movingon to the elution step.

The first purification procedure 500 can also comprise introducing water512 (e.g., deionized water) to the first reaction vessel 502 to eluteamplicons bound to the magnetic beads 504. For example, the purificationprocedure 500 can comprise introducing about 20 μL of deionized water tothe first reaction vessel 502 to elute the amplicons bound to themagnetic beads 504. The first purification procedure 500 can furthercomprise removing a first amplicon-containing supernatant 514 from thefirst reaction vessel 402 after the introduction of water 512 while themagnetic beads 504 are immobilized to the at least one inner surface ofthe first reaction vessel 402 by the magnet 508. For example, the firstamplicon-containing supernatant 514 can be aspirated from the firstreaction vessel 402 using a micropipette and transferred to anintermediary reaction vessel 516.

The aforementioned purification steps can then be repeated again usingcontents within the intermediary reaction vessel 516. For example, thefirst purification procedure 500 can further comprise introducingadditional instances of the magnetic bead suspension 502 to theintermediary reaction vessel 516 and incubating a mixture within theintermediary reaction vessel 516 comprising both the firstamplicon-containing supernatant 514 and the magnetic bead suspension 502at room temperature (e.g., between about 20° C. to about 25° C.) for anincubation period (e.g., between about 5 minutes to 10 minutes) to allowthe target amplicons to bind to the magnetic beads 504.

The first purification procedure 500 can also comprise collecting andimmobilizing the amplicon-bound magnetic beads to at least one innersurface of the intermediary reaction vessel 516 by placing at least oneouter surface of the intermediary reaction vessel 516 in proximity to amagnet 508. The first purification procedure 500 can also compriseremoving and discarding a supernatant from the intermediary reactionvessel 516 while the amplicon-bound magnetic beads are immobilized tothe at least one inner surface of the intermediary reaction vessel 516by the magnet 508.

The first purification procedure 500 can also comprise introducing anethanol wash solution 510 to the intermediary reaction vessel 516containing the amplicon-bound magnetic beads. The first purificationprocedure 500 can further comprise removing and discarding a supernatantcomprising primarily of the ethanol wash solution 510 from theintermediary reaction vessel 516 while the amplicon-bound magnetic beadsare immobilized. The first purification procedure can also comprisedrying (e.g., air drying) the intermediary reaction vessel 516 aftereach ethanol wash to evaporate the ethanol left over. The ethanol washsteps can be repeated one or more times in succession. For example, theethanol wash steps can be performed twice before moving on to theelution step. The first purification procedure 500 can also compriseintroducing water 512 (e.g., deionized water) to the intermediaryreaction vessel 516 to elute target amplicons bound to the magneticbeads 504.

The first purification procedure 500 can further comprise removing asecond amplicon-containing supernatant from the intermediary reactionvessel after the introduction of the water while the magnetic beads 504are immobilized to at least one inner surface of the intermediaryreaction vessel 514 by the magnet 508. The second amplicon-containingsupernatant removed from the intermediary reaction vessel is thepurified target amplicon solution 518.

Although FIG. 5 illustrates the first reaction vessel 402 and theintermediary reaction vessel 516 as standalone reaction tubes or PCRtubes, it is contemplated by this disclosure that any of the firstreaction vessel 402 or the intermediary reaction vessel 516 can be awell of a multi-well plate (e.g., a multi-well PCR plate), such as a96-well plate or a 384-well plate.

FIG. 6A illustrates an embodiment of a magnetic separation rack 600comprising a plurality of wells 602 with at least one magnet 508positioned at the bottom of each well 602. For example, the magneticseparation rack 600 can be a DynaMag® magnetic rack for holdingnon-skirted or semi-skirted 96-well or 384-well PCR plates. In otherembodiments, the magnetic separation rack 600 can be any type ofmagnetic rack or platform comprising one or more magnets 508 positionedon the bottom or sides of the rack or platform. The magnets 508 of themagnetic separation rack 600 can aggregate and collect the magneticbeads 504 including the amplicon-bound magnetic beads 507.

FIG. 6A illustrates that at least part of a reaction vessel (e.g., thefirst reaction vessel 402) can be positioned into a well 602 of themagnetic separation rack 600 in proximity to a magnet 508 at the bottomof the well. In some embodiments, the reaction vessel can be lifted outof the well 602 and then placed back into the well 602. This can berepeated until the magnetic beads 504 gather (e.g., as a pellet or inpellet form) at the bottom of the reaction vessel 402. The magneticbeads 504 within the reaction vessel can be immobilized when thereaction vessel 402 is supported upright by the well 602 and at leastpart of the reaction vessel is positioned within the well 602.

Although FIG. 6A illustrates an instance of the reaction vessel as asingular reaction tube or PCR tube, it is contemplated by thisdisclosure that the reaction vessel can refer to one well of amulti-well plate (e.g., a well of a 96-well PCR plate) and the entiremulti-well plate can be positioned on the magnetic separation rack 600such that each of the wells of the multi-well plate is positioned withina well 602 of the magnetic separation rack.

FIG. 6B illustrates another embodiment of a magnetic separation rack604. The magnetic separation rack 604 shown in FIG. 6B can comprisemagnets 508 designed as magnetic bar 606 or columnar-type magnetsextending (e.g., perpendicularly or angularly) from a bottom surface ofthe rack. The magnetic separation rack 604 shown in FIG. 6B can bedesigned for use with skirted multi-well plates (e.g., a skirted 96-wellor 384-well PCR plate). For example, the magnetic separation rack 604can be a DynaMag® side skirted magnetic rack for holding skirted 96-wellor skirted 384-well PCR plates.

FIG. 6C illustrates an embodiment of a skirted multi-well plate 608(e.g., a skirted 96-well or 384-well PCR plate) positioned on themagnetic separation rack 604 of FIG. 6B. At least one column of wells610 of the multi-well plate 608 can be positioned next to or inproximity to a magnetic bar 606 of the skirted multi-well plate 608. Tocollect and immobilize the magnetic beads 504 (including theamplicon-bound magnetic beads), the skirted multi-well plate 608 can beshifted laterally left-to-right and vice versa such that the column ofwells 610 is brought close to the magnetic bar 606, briefly moved awayfrom the magnetic bar 606, and then brought back next to the magneticbar 606. This can be repeated until the magnetic beads 504 gather oraccumulate (e.g., as a pellet or in pellet form) near an inner sidesurface of the wells 610.

In this embodiment, the individual wells (or individual reactionvessels) of the multi-well plate 608 can have at least one outer surfaceof the well positioned next to the magnetic bar 606, briefly moving orshifting the well away from the magnetic bar 606, and then bringing thewell back next to the magnetic bar 606. The magnetic beads 504 withinthe column of wells 610 can be immobilized when the column of wells 610is positioned next to the magnetic bar 606.

Although FIG. 6C illustrates a skirted multi-well plate, it iscontemplated by this disclosure that the magnetic separation rack 604 ofFIG. 6B can also be used with non-skirted multi-well plates orsemi-skirted multi-well plates. Moreover, although FIG. 6C illustrates amulti-well plate, the reaction vessel can also be a singular reactiontube or PCR tube or a plurality of such tubes held by clamps, roboticarms, or other types of holders (e.g., in a column or row), and thesingular reaction tube or the plurality of tubes can be positioned closeto a magnetic bar 606 and then repeatedly shifted away from and backtoward the magnetic bar 606 until the magnetic beads 504 are collectedand immobilized within the singular reaction tube or tubes. The magneticbeads 504 can be collected and immobilized to an inner side surface ofthe singular reaction tube when an outer side surface of the singularreaction tube is positioned in proximity to or next to the magnetic bar606.

FIG. 6D is a black-and-white image illustrating an embodiment of a wellplate 612 having magnetic beads 504 immobilized to the inner sidesurfaces of wells of the well plate 612. As shown in FIG. 6D, the wellplate 612 can be a semi-skirted well plate such as a semi-skirted96-well plate The wells of the well plate 612 can serve as reactionvessels (e.g., the first reaction vessel 402) for undergoing certainsteps of the first purification procedure 500 using the magnetic beads504.

The magnetic separation racks 600 shown in FIGS. 6A-6D can be used aspart of the first purification procedure 500, a second procedure 800(see FIG. 8), or a combination thereof. For example, any of the firstreaction vessel 402 and the second reaction vessel 700 can be positionedon the magnetic separation rack 600 in order to collect and immobilizethe magnetic beads 504 within such reaction vessels.

FIG. 7 illustrates that an aliquot of the purified target ampliconsolution 518 can be transferred to a second reaction vessel 700comprising a second reagent mixture 702 to yield a second reactionmixture 704.

In some embodiments, the second reaction vessel 700 can be a single PCRreaction tube 408 or vessel. In other embodiments, the second reactionvessel 700 can be one well 410 of a multi-well plate 412 (e.g., amulti-well PCR plate), such as a 96-well plate or a 384-well plate.

In some embodiments, transferring the aliquot of the purified targetamplicon solution 518 into the second reaction vessel 700 containing thesecond reagent mixture 702 can comprise transferring about 10.5 μL ofthe purified target amplicon solution 518 into the second reactionvessel 700 containing about 14.5 μL of the second reagent mixture 702.The aliquot of the purified target amplicon solution 518 can betransferred using a pipette such as a fixed-volume micropipette or anadjustable-volume micropipette.

The second reagent mixture 702 can comprise a reagent solution and anindex primer pool comprising a plurality of index adapteroligonucleotides or index adapters. The reagent solution can comprise aTaq DNA polymerase, a plurality of dNTPs, a cofactor, a nonionicsurfactant, a gelatin solution, a glycerol solution, and one or morereagent buffers.

The index adapters can be annealed or added to the ends of the amplifiedtarget sequences (or target amplicons) within the purified targetamplicon solution after the second PCR protocol. The index adapters whenadded to the ends of the target amplicons can act as barcodes or uniqueidentifiers to identify the target amplicons when the DNA library isbeing sequenced using next-generation sequencing. Once the targetamplicons are tagged with the index adapters, the DNA library can beconsidered ready for sequencing using next-generation sequencing systemssuch as the Illumina MiSeq® system. Different pairs of index adapterscan also be used to allow multiple pooled samples to be sequencedtogether in a single high-throughput next-generation sequencing run.

In some embodiments, the index adapters can be overhang adapters. Forexample, the index adapters can be Nextera® XT index primers provided byIllumina, Inc. and compatible with Illumina's MiSeq® next-sequencingsystem. As a more specific example, the index adapters can compriseNextera® XT index 1 primers (with P7 adapters) and Nextera® XT index 2primers (with P5 adapters).

FIG. 8 illustrates an embodiment of the second purification procedure800. The second purification procedure 800 can comprise introducing amagnetic bead suspension 502 to the second reaction vessel 700 after thesecond PCR protocol. The magnetic bead suspension 502 can comprisemagnetic beads 504 configured to allow the index-tagged amplicons withinthe amplified second reaction mixture 704 to selectively bind tosurfaces of the magnetic beads 504. For example, the magnetic beadsuspension 502 can be AMPure® beads manufactured by Beckman Coulter,Inc.

In some embodiments, the volume of the magnetic bead suspension 502added is anywhere between 1× to 1.8× the volume of the second reactionmixture 704 within the second reaction vessel 700. For example, 36 μL ofthe magnetic bead suspension 502 can be added to 20 μL of the secondreaction mixture 704 within the second reaction vessel 700.

The second purification procedure 800 can also comprise incubating amixture 802 within the second reaction vessel 700 comprising both theamplified second reaction mixture 704 and the magnetic bead suspension502 at room temperature (e.g., between about 20° C. to about 25° C.) foran incubation period (e.g., between about 5 minutes to 10 minutes) toallow the index-tagged target amplicons to bind to the magnetic beads504.

The second purification procedure 800 can also comprise collecting andimmobilizing the amplicon-bound magnetic beads 804 to at least one innersurface of the second reaction vessel 700 by placing at least one outersurface of the second reaction vessel 700 in proximity to a magnet 508.The second purification procedure 800 can comprise initially positioningthe at least one outer surface of the second reaction vessel 700 inproximity to the magnet 508 and then repeatedly moving the secondreaction vessel 700 away from the magnet 508 and bringing the at leastone outer surface of the second reaction vessel 700 back next to themagnet 508.

In some embodiments, the magnet 508 can be a permanent magnet. Forexample, the magnet 508 can be a neodymium iron boron (NdFeB) permanentmagnet. The magnet 508 can be incorporated into or embedded within amagnetic separation rack or platform (see, e.g., FIGS. 6A and 6B).

The second purification procedure 800 can also comprise removing anddiscarding a supernatant from the second reaction vessel 700 while theamplicon-bound magnetic beads 804 are immobilized to the at least oneinner surface of the second reaction vessel 700 by the magnet 508.Removing and discarding the supernatant can comprise using amicropipette to aspirate the supernatant from the second reaction vessel700 into the pipette tip and expelling the supernatant to discard thesupernatant.

The second purification procedure 800 can also comprise introducing anethanol wash solution 510 to the second reaction vessel 700 containingthe amplicon-bound magnetic beads 804. For example, the ethanol washsolution 510 can be a 70% (v/v) ethanol or isopropyl alcohol solution.The second purification procedure 800 can comprise introducing betweenabout 50 μL to about 125 μL of the ethanol wash solution 510 to thesecond reaction vessel 700 containing the amplicon-bound magnetic beads507. One objective of the ethanol wash step is to remove excess saltsfrom buffers added to the second reaction vessel 700 in previous stepsof the method 200.

The second purification procedure 800 can further comprise removing anddiscarding a supernatant comprising primarily of the ethanol washsolution 510 from the second reaction vessel 700 while theamplicon-bound magnetic beads 804 are immobilized to the at least oneinner surface of the second reaction vessel 700 by the magnet 508. Thesecond purification procedure 800 can also comprise drying (e.g., airdrying) the second reaction vessel 700 after each ethanol wash toevaporate the ethanol left over. The ethanol wash steps can be repeatedone or more times in succession. For example, the ethanol wash steps canbe performed twice before moving on to the elution step.

The second purification procedure 800 can also comprise introducingwater 512 (e.g., deionized water) to the second reaction vessel 700 toelute amplicons bound to the magnetic beads 504. For example, the secondpurification procedure 800 can comprise introducing about 20 μL ofdeionized water to the second reaction vessel 700 to elute the ampliconsbound to the magnetic beads 504. The second purification procedure 800can further comprise removing a first amplicon-containing supernatant806 from the second reaction vessel 700 after the introduction of water512 while the magnetic beads 504 are immobilized to the at least oneinner surface of the second reaction vessel 700 by the magnet 508. Forexample, the first amplicon-containing supernatant 806 can be aspiratedfrom the second reaction vessel 700 using a micropipette and transferredto an intermediary reaction vessel 808.

The aforementioned purification steps can then be repeated again usingcontents within the intermediary reaction vessel 808. For example, thesecond purification procedure 800 can further comprise introducingadditional instances of the magnetic bead suspension 502 to theintermediary reaction vessel 808 and incubating a mixture within theintermediary reaction vessel 808 comprising both the firstamplicon-containing supernatant 806 and the magnetic bead suspension 502at room temperature (e.g., between about 20° C. to about 25° C.) for anincubation period (e.g., between about 5 minutes to 10 minutes) to allowthe index-tagged target amplicons to bind to the magnetic beads 504.

The second purification procedure 800 can also comprise collecting andimmobilizing the amplicon-bound magnetic beads to at least one innersurface of the intermediary reaction vessel 808 by placing at least oneouter surface of the intermediary reaction vessel 808 in proximity to amagnet 508. The second purification procedure 800 can also compriseremoving and discarding a supernatant from the intermediary reactionvessel 808 while the amplicon-bound magnetic beads are immobilized tothe at least one inner surface of the intermediary reaction vessel 808by the magnet 508.

The second purification procedure 800 can also comprise introducing anethanol wash solution 510 to the intermediary reaction vessel 808containing the amplicon-bound magnetic beads. The second purificationprocedure 800 can further comprise removing and discarding a supernatantcomprising primarily of the ethanol wash solution 510 from theintermediary reaction vessel 808 while the amplicon-bound magnetic beadsare immobilized. The second purification procedure 800 can also comprisedrying (e.g., air drying) the intermediary reaction vessel 808 aftereach ethanol wash to evaporate the ethanol left over. The ethanol washsteps can be repeated one or more times in succession. For example, theethanol wash steps can be performed twice before moving on to theelution step. The second purification procedure 800 can also compriseintroducing water 512 (e.g., deionized water) to the intermediaryreaction vessel 808 to elute the index-tagged target amplicons bound tothe magnetic beads 504.

The second purification procedure 800 can further comprise removing asecond amplicon-containing supernatant from the intermediary reactionvessel after the introduction of the water while the magnetic beads 504are immobilized to at least one inner surface of the intermediaryreaction vessel 514 by the magnet 508. The second amplicon-containingsupernatant removed from the intermediary reaction vessel is thepurified index-tagged library 810 that can be sequenced using anext-generation sequencing protocol such as an Illumina® NGS protocol,an Ion Personal Genome Machine® (PGM) protocol, a SOLiD® NGS protocol,or a combination thereof.

Although FIG. 8 illustrates the second reaction vessel 700 and theintermediary reaction vessel 808 as standalone reaction tubes or PCRtubes, it is contemplated by this disclosure that any of the secondreaction vessel 700 or the intermediary reaction vessel 808 can be awell of a multi-well plate (e.g., a multi-well PCR plate), such as a96-well plate or a 384-well plate.

FIG. 9 illustrates the size distribution of a 16S DNA library prepareddirectly from a stool sample using the method 200 disclosed herein. The16S DNA library can be analyzed using a bioanalyzer kit or system suchas an Agilent® bioanalyzer chip. As previously discussed, the 16S primerpool of the first reaction mixture 406 contained 16S forward and reverseprimers targeting the V3 and V4 variable regions of the 16S rRNA gene.Amplicons comprising the V3 and V4 variable regions are expected to havea size of about 500 bp to about 600 bp. As shown in FIG. 9, the 16S DNAlibrary analyzed comprised a significant amount of amplicons of thissize when the lower markers (˜15 bp) and upper markers (1500 bp) usedfor the alignment and quantitation of the DNA library by the bioanalyzerare discounted.

FIGS. 10A to 10C illustrate comparisons of 16S DNA libraries preparedfrom three different stool samples using traditional DNA extractionmethods with a QIAmp® DNA Stool Mini Kit (Cat. No. 51504) and 16S DNAlibraries prepared from the same three stool samples using the directamplification method 200 disclosed herein. As shown in FIGS. 10A to 10C,the method 200 worked at least as well as traditional extraction methodsin isolating DNA from bacteria from different taxonomic groups.

Also, important to note here is that the DNA libraries (e.g., the 16SDNA libraries) prepared using the method 200 disclosed herein were eachprepared in a shorter period of time than libraries prepared usingtraditional DNA extraction methods. Moreover, the DNA libraries preparedusing the method 200 disclosed herein did not require the user topurchase expensive extraction kits and one-time use spin columns.

Each of the individual variations or embodiments described andillustrated herein has discrete components and features which may bereadily separated from or combined with the features of any of the othervariations or embodiments. Modifications may be made to adapt aparticular situation, material, composition of matter, process, processact(s) or step(s) to the objective(s), spirit or scope of the presentinvention.

Methods recited herein may be carried out in any order of the recitedevents that is logically possible, as well as the recited order ofevents. Moreover, additional steps or operations may be provided orsteps or operations may be eliminated to achieve the desired result.

Furthermore, where a range of values is provided, every interveningvalue between the upper and lower limit of that range and any otherstated or intervening value in that stated range is encompassed withinthe invention. Also, any optional feature of the inventive variationsdescribed may be set forth and claimed independently, or in combinationwith any one or more of the features described herein.

All existing subject matter mentioned herein (e.g., publications,patents, patent applications and hardware) is incorporated by referenceherein in its entirety except insofar as the subject matter may conflictwith that of the present invention (in which case what is present hereinshall prevail). The referenced items are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such material by virtue of prior invention.

Reference to a singular item, includes the possibility that there areplural of the same items present. More specifically, as used herein andin the appended claims, the singular forms “a,” “an,” “said” and “the”include plural referents unless the context clearly dictates otherwise.It is further noted that the claims may be drafted to exclude anyoptional element. As such, this statement is intended to serve asantecedent basis for use of such exclusive terminology as “solely,”“only” and the like in connection with the recitation of claim elements,or use of a “negative” limitation. Unless defined otherwise, alltechnical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisinvention belongs.

In understanding the scope of the present disclosure, the term“comprising” and its derivatives, as used herein, are intended to beopen-ended terms that specify the presence of the stated features,elements, components, groups, integers, and/or steps, but do not excludethe presence of other unstated features, elements, components, groups,integers and/or steps. The foregoing also applies to words havingsimilar meanings such as the terms, “including”, “having” and theirderivatives. Also, the terms “part,” “section,” “portion,” “member”“element,” or “component” when used in the singular can have the dualmeaning of a single part or a plurality of parts. As used herein, thefollowing directional terms “forward, rearward, above, downward,vertical, horizontal, below, transverse, laterally, and vertically” aswell as any other similar directional terms refer to those positions ofa device or piece of equipment or those directions of the device orpiece of equipment being translated or moved. Finally, terms of degreesuch as “substantially”, “about” and “approximately” as used herein meana reasonable amount of deviation (e.g., a deviation of up to ±5%) of themodified term such that the end result is not significantly ormaterially changed.

This disclosure is not intended to be limited to the scope of theparticular forms set forth, but is intended to cover alternatives,modifications, and equivalents of the variations or embodimentsdescribed herein. Further, the scope of the disclosure fully encompassesother variations or embodiments that may become obvious to those skilledin the art in view of this disclosure.

1. A method of preparing a deoxyribonucleic acid (DNA) library from astool sample for downstream next-generation sequencing, comprising:applying a stool sample directly to a buffer solution; heating thebuffer solution containing the stool sample to a temperature of 90° C.to 100° C. and cooling the buffer solution containing the stool sampleto room temperature of 20° C. to 25° C.; separating a supernatant withinthe buffer solution containing the stool sample from a precipitate usingcentrifugation when the buffer solution containing the stool sample hasreached the room temperature of 20° C. to 25° C. and transferring analiquot of the supernatant into a first reaction vessel containing afirst reagent mixture to yield a first reaction mixture, wherein thefirst reagent mixture comprises: Taq DNA polymerase, dNTPs, a primerpool comprising a plurality of forward primers and reverse primers,magnesium chloride (MgCl₂), a nonionic surfactant, a gelatin solution, aglycerol solution, and a buffer solution; subjecting the first reactionmixture in the first reaction vessel to a first polymerase chainreaction (PCR) protocol; purifying the first reaction mixture within thefirst reaction vessel through a first purification procedure using amagnetic bead suspension, and multiple washes using an ethanol washsolution, and water as an eluent to yield a purified target ampliconsolution, wherein the first purification procedure further comprises:(a) introducing the magnetic bead suspension to the first reactionvessel, wherein magnetic beads within the magnetic bead suspension areconfigured to allow amplicons within the amplified first reactionmixture to selectively bind to surfaces of the magnetic beads; (b)incubating a mixture within the first reaction vessel comprising themagnetic bead suspension at 20° C. to 25° C. for an incubation period toallow the amplicons to bind to the magnetic beads within the magneticbead suspension; (c) collecting and immobilizing the amplicon-boundmagnetic beads to at least one inner surface of the first reactionvessel by placing at least one outer surface of the first reactionvessel in proximity to a magnet, wherein the first reaction vessel is awell of a multi-well plate and the magnet is part of a magneticseparation rack or platform and wherein collecting and immobilizing theamplicon-bound magnetic beads further comprises positioning the at leastone outer surface of the first reaction vessel next to the magnet, andwherein the method further comprises an additional step of moving thefirst reaction vessel away from the magnet and bringing the at least oneouter surface of the first reaction vessel back next to the magnet; (d)removing and discarding a supernatant from the first reaction vesselwhile the amplicon-bound magnetic beads are immobilized to the at leastone inner surface of the first reaction vessel by the magnet; (e)introducing an ethanol wash solution to the first reaction vesselcomprising the amplicon-bound magnetic beads; (f) removing anddiscarding a wash supernatant from the first reaction vessel while theamplicon-bound magnetic beads are immobilized to the at least one innersurface of the first reaction vessel by the magnet; (g) introducingwater to the first reaction vessel to elute amplicons bound to themagnetic beads; (h) removing a first amplicon-containing eluate from thefirst reaction vessel after the introduction of water while the magneticbeads are immobilized to the at least one inner surface of the firstreaction vessel by the magnet; (i) adding the first amplicon-containingeluate from step (h) to an intermediary reaction vessel and repeatingsteps (a) through (g) using contents within the intermediary reactionvessel; and (j) removing a second amplicon-containing eluate from theintermediary reaction vessel after the introduction of the water whilethe magnetic beads are immobilized to at least one inner surface of theintermediary reaction vessel by the magnet, wherein the secondamplicon-containing supernatant removed is the purified target ampliconsolution; transferring the purified target amplicon solution to a secondreaction vessel comprising a second reagent mixture to yield a secondreaction mixture, wherein the second reagent mixture comprises: Taq DNApolymerase, dNTPs, a plurality of forward and reverse primers comprisingindex adapter oligonucleotides, magnesium chloride (MgCl₂), a nonionicsurfactant, a gelatin solution, a glycerol solution, and a buffersolution; subjecting the second reaction mixture in the second reactionvessel to a second PCR protocol; purifying index-tagged amplicons withinthe second reaction vessel through a second purification procedure usingadditional instances of the magnetic bead suspension and multiple washesusing additional instances of the ethanol wash solution and water as aneluent to yield a purified index-tagged DNA library, wherein thepurified index-tagged DNA library is ready for downstreamnext-generation sequencing.
 2. (canceled)
 3. (canceled)
 4. (canceled) 5.The method of claim 1, wherein the second purification procedure furthercomprises: (a) introducing additional instances of the magnetic beadsuspension to the second reaction vessel, wherein the magnetic beadswithin the magnetic bead suspension are configured to allow ampliconswithin the amplified second reaction mixture to selectively bind tosurfaces of the magnetic beads; (b) incubating a mixture within thesecond reaction vessel comprising the magnetic bead suspension at 20° C.to 25° C. for an incubation period to allow amplicons to bind to beadswithin the magnetic bead suspension; (c) collecting and immobilizing theamplicon-bound magnetic beads to at least one inner surface of thesecond reaction vessel by placing at least one outer surface of thesecond reaction vessel in proximity to a magnet; (d) removing anddiscarding a supernatant from the second reaction vessel while theamplicon-bound magnetic beads are immobilized to the at least one innersurface of the second reaction vessel by the magnet; (e) introducing anethanol wash solution to the second reaction vessel comprising theamplicon-bound magnetic beads; (f) removing and discarding a washsupernatant from the second reaction vessel while the amplicon-boundmagnetic beads are immobilized to the at least one inner surface of thesecond reaction vessel by the magnet; (g) introducing water to thesecond reaction vessel to elute amplicons bound to the magnetic beads;(h) removing a first amplicon-containing eluate from the second reactionvessel after the introduction of the water while the magnetic beads areimmobilized to the at least one inner surface of the second reactionvessel by the magnet; (i) adding the first amplicon-containing eluatefrom step (h) to another intermediary reaction vessel and repeatingsteps (a) through (g) using contents within the other intermediaryreaction vessel; and (j) removing a second amplicon-containing eluatefrom the other intermediary reaction vessel after the introduction ofthe water while the magnetic beads are immobilized to at least one innersurface of the other intermediary reaction vessel by the magnet, whereinthe second amplicon-containing supernatant removed is the purifiedindex-tagged DNA library.
 6. The method of claim 1, wherein the firstPCR protocol comprises the steps of: (i) heating the first reactionmixture to activate the Taq DNA polymerase in an activation step; (ii)further heating the first reaction mixture to denature nucleic acidswithin the first reaction mixture; (iii) lowering the temperature toallow for annealing and extension, (iv) repeating steps (ii) and (iii)for at least 4 more cycles, (v) further heating the first reactionmixture to further denature nucleic acids within the first reactionmixture; (vi) lowering the temperature to allow for annealing andextension, (vii) repeating steps (v) and (vi) for at least 24 morecycles, and (viii) holding the amplified first reaction mixture withinthe first reaction vessel at a holding temperature.
 7. The method ofclaim 6, wherein the second PCR protocol comprises the steps of: (i)heating the second reaction mixture to activate the Taq DNA polymerasein an activation step; (ii) further heating the second reaction mixtureto denature nucleic acids within the reaction mixture; (iii) loweringthe temperature to allow for annealing and extension, (iv) raising thetemperature to allow for additional extension, (v) repeating steps (ii)through (iv) for between 7 cycles and 9 cycles, (vi) further heating thesecond reaction mixture to allow for further extension, and (vii)holding the amplified second reaction mixture within the second reactionvessel at a holding temperature, wherein the amplified second reactionmixture is ready for further purification.
 8. The method of claim 1,wherein applying the stool sample directly to the buffer solutionfurther comprises applying between 3 mg to 10 mg of the stool sample to100 μL of the buffer solution and wherein transferring the aliquot ofthe supernatant into the first reagent mixture in the first reactionvessel further comprises transferring 2 μL of the supernatant into thefirst reagent mixture in the first reaction vessel.
 9. The method ofclaim 1, wherein the primer pool comprises a plurality of 16S forwardprimers and 16S reverse primers for targeting variable regions V3 and V4of the 16S ribosomal ribonucleic acid (rRNA) gene.
 10. The method ofclaim 1, wherein the first and second reagent mixture further comprise atris(hydroxymethyl)aminomethane (Tris)-hydrochloric acid (HCl) buffersolution and a potassium chloride (KCl) buffer solution, and wherein thenonionic surfactant is a polysorbate 20 solution. 11.-20. (canceled)